We wish to understand the adhesive and signaling functions of the transmembrane protein integrin in the context of intact animal embryos. Integrins are involved directly in many fundamental biological processes, and have been implicated in diseases ranging from muscular dystrophies to cancers. This proposal focuses on integrin signaling, and specifically upon the recently discovered signaling molecule integrin-linked kinase (ILK), Overexpression of ILK in cultured cells alters cellular adhesive properties, induces adhesion-independent growth, induces tumorigenicity, and also stimulates fibronectin assembly in the extracellular matrix, There are no reported ILK mutants to date, so relatively little is known about ILK function in vivo. The experiments in this proposal follow up the recent discovery that the C. elegans pat-4 gene codes for an ILK homolog. Existing pat-4 mutants will be used to directly investigate ILK function during muscle development in vivo. Specific aims: 1. Are transcripts of pat-4 alternatively spliced? cDNA sequence analysis and reverse-transcription PCR will be performed, and may reveal novel ILK isoforms. 2. Which ILK domains are altered by the existing pat-4 mutations? All four existing pat-4 mutant alleles will be characterized by DNA sequence analysis. 3. Is the PAT-4 protein located in focal adhesion-like muscle cell attachments call dense bodies, and related attachments called M-lines? PAT-4 will be localized in embryos and adults using a) antisera and b) PAT 4::Green Fluorescent Protein (GFP) chimeric proteins. 4. Is PAT-4 ILK needed for attachment structure and extracellular matrix (ECM) assembly in vivo? The defective assembly of attachment structures in pat-4 loss-of-function mutants will be analyzed in detail through use of a large, existing collection of antibodies and GFP-tagged components. 5. What are the requirements for proper ILK localization to nascent muscle cell attachment structures? Attachment structure assembly will be blocked at different stages using existing mutations in various component genes, and the assembly of PAT-4 assayed. 6. Are the kinase domain, phosphoinositide lipid-binding motif, and/or ankyrin repeats of ILK needed to regulate dense body and M-line assembly? PAT -4 will be altered by site-directed mutagenesis and then tested in transgenic animals. Deletion of ankyrin repeats will test their hypothetical function in recruiting the recently discovered LIM domain protein UNC-97/PINCH to nascent attachments. 7. Is PAT-4 needed for normal myoblast proliferation, migration, and insertion in vivo? Time-lapse analysis of myogenesis will be performed on wild-type and mutant embryos expressing GFP-tagged proteins.